The proposed research will evaluate the role in cellular aging of lipid peroxidation, proposed to be of central importance in aging by the free radical theory. Normal human and bovine adrenocortical cells, which show retention of differentiated functions throughout a finite life span in culture, will be used to evaluate the effects of mitochondrial lipid peroxidation on a variety of cellular functions. The mitochondria of adrenocortical cells contain cytochrome P-450 11 beta which autoxidizes in the presence of 11 beta-hydroxylated steroid pseudosubstrates with initiation of lipid peroxidation. The effects of this lipid peroxidation on various cellular functions will be examined. The products of lipid peroxidation will be detected after extraction and high performance liquid chromatographic separation. Quantitation of lipid peroxides and fluorescent products of lipid peroxidation will be carried out. Effects on mitochondrial steroid metabolizing cytochrome P-450 activities, particularly the aldosterone-synthesizing system, as well as 11 beta-hydroxylase, and on other mitochondrial functions, particularly preference for 2-oxoglutarate from glutamine rather than pyruvate as tricarboxylic acid cycle substrate, will be determined. Drug-metabolizing cytochrome P-450 activities will be examined. Effects on cell membrane receptor-associated functions, including ACTH-stimulated cAMP production and angiotensin II- and FGF-stimulated DNA synthesis, and on cell culture life span, cloning efficiency, and prostaglandin synthesis will be determined. There is evidence that all these cellular functions may be affected by lipid peroxidation. In each case, the effects of antioxidants and lowered oxygen concentration will be evaluated. Antioxidants and low oxygen may allow maintenance of a varety of cellular differentiated functions over an extended life span in normal cultured human cells. This will allow evaluation of the significance of mitochondrial lipid peroxidation in normal function and its relevance to the cellular aging process.